This invention relates to a method for generating large numbers of fertilizable ova from the ovarian tissue of a mammal. More specifically, it relates to methods for the in vitro development and maturation of germ cells recovered from the ovarian tissue of a mammal into fertilizable oocytes. These oocytes may be fertilized in vitro to form embryos which can be implanted into surrogate mothers.
The two commonly employed methods for breeding animals are natural mating and artificial insemination. In addition, a small number of animals are bred by embryo transfer. In this technique, a female is impregnated either naturally or by artificial insemination and six to ten days thereafter her uterus is flushed and any viable embryos are recovered. The recovered embryos are then implanted into surrogate mothers. Often the female is superovulated by hormone treatments prior to impregnation to increase the yield of embryos. For example, in cows, superovulation can increase the number of viable embryos recovered from one to about five to fifteen.
With the widespread acceptance and use of artificial insemination for animal breeding, large quantities of sperm are commonly collected and banked for future use, in essence creating a limitless supply of sperm. Equally important, artificial insemination permits the genes from the most desirable animals to be made far more widely available than with natural mating techniques. For example, sperm collected from a single superior bull can be used to impregnate thousands of cows by artificial insemination. Heretofore, however, there has existed no practical method for collecting large numbers of fertilizable ova from females and, as a result, there is no efficient method comparable to artificial insemination for improving the quality of livestock by the widespread breeding of superior females. Embryo transfer which is designed to overcome this problem is inefficient and expensive and therefore not appropriate for widespread use.
The reason large numbers of fertilizable ova have heretofor not been available relates to the reproductive biology of females. Mature males are continuously producing large numbers of sperm. However, in female mammals, only certain cells in the ovaries are capable of maturing into ova. These germ cells, which usually number about 200,000 to 300,000 per ovary in most mammals, are present at birth, are held in the ovary, arrested in an early stage of meiosis and incapable of being fertilized and developing into normal young. Under normal circumstances, a number of these cells begin to develop within the ovary with a periodicity tuned to the animal's sexual cycle. At the appropriate time in the sex cycle, either one or a small number of these cells (depending on whether the animal is a litter bearer) will be released from the ovary, a process known as ovulation. The complex process by which an individual germ cell develops to the point at which ovulation occurs is known as folliculogenesis. Folliculogenesis involves several major steps and the coordinated activities of other cells of the ovary as well as pituitary and ovarian hormones. Following ovulation, the cell or cells (oocytes) released undergo another step in the process of meiosis in which the number of chromosomes in the cells is reduced by half, after which cells become fertilizable ova. This process usually occurs during the movement of the cells from the ovary to the oviduct where fertilization will take place.
To the knowledge and belief of the inventors, there currently exists no known method or process for the development and maturation of large numbers of germ cells into fertilizable ova either in vitro or in vivo. There is a clear need for such a process in order to eliminate the critical limiting factor in all known breeding techniques, the availability of fertilizable ova. Such a process would make possible the use of in vitro fertilization and embryo implantation on a large scale for efficiently reproducing commercially important animals or other important animals (such as endangered species). Perhaps more importantly, it would also make possible the use of these techniques for efficiently improving the quality of herds and of animals by selectively breeding desirable females.